Angiogenesis of tumors is a major threat to cancer patients as it enables further tumor growth and facilitates metastasis. Angiogenesis occurs by extension of branched protrusions of endothelial cells into 3 dimensional (3D) extracellular space, similar to the branching morphogenesis of neurons. To identify molecular pathways implicated in this kind of morphogenesis, and to test methods to intervene in angiogenesis, novel quantitative tools are needed that measure this process in spatial and temporal resolution commensurate with its molecular regulation. Towards this end, we propose to develop a "morphodynamic profile" (MP) methodology that tracks cell surface movements in 3D and classifies them by statistical analysis of protrusion and retraction patterns. Our long term goal is to exploit these readouts as a quantitative platform to establish the functional relationships between actin cytoskeleton regulation and angiogenic sprouting of endothelial cells. To develop this methodology, we propose to: (1) optimize our existing 3D endothelial culture system and microscopy set up for high resolution, live cell image collection; (2) develop computer algorithms for complete and automated cell surface tracking and for statistical analysis and visualization of protrusion patterns for phenotypic characterization of control cells versus cells expressing mutations in molecules implicated in the regulation of branch morphogenesis; and (3) demonstrate the sensitivity of our profiling methodology by classifying the effects of rac1 and cofilin mutants, and measure the MP differences caused by their expression. The completion of this proposal will enable future work by us, and the scientific community at large, to quantitatively investigate the mechanisms behind angiogenesis and pathfinding in 3D. The purpose of this proposal is to develop quantitative image analysis tools to better our understanding of cell migration in three dimensional environments. In particular, we will focus on analyzing branching morphogenesis, a key process in angiogenesis and neuronal pathfinding. [unreadable] [unreadable] [unreadable]